A synchronized heart beat is controlled by pace-making signals of the cardiac conduction system (CCS). Future therapeutic approaches to regeneration or repair of this essential heart tissue after injury or congenital disease would benefit from a clear understanding of the mechanisms that regulate differentiation of the CCS. This project aims to determine for the first time the molecular signals that induce differentiation of the CCS in vivo. Our studies in the embryonic chick heart have shown that: 1) impulse- conducting Purkinje cells differentiate from myocytes during embryogenesis; 2) this conversion of contractile myocytes into conduction cells is induced by local signals derived from developing coronary arteries in vivo; 3) the vascular cytokine, endothelin (ET), induces embryonic myocytes to differentiate into Purkinje cells in vitro; 4) ET-induced Purkinje fiber differentiation is mediated by binding of ET to its G protein-coupled receptors (ETRs) ubiquitously expressed by all myocytes; and 5) Purkinje fiber differentiation is triggered only when ET is proteolyticaly activated from its precursor, big-ET, by the ET-converting enzyme (ECE). Therefore, we hypothesize that a) the ET-signal is a local, instructive cue that recruits myocytes to Purkinje cells; and b) activation of ET-signaling is spatially and temporally confined by ECE-expression in the embryonic heart. In the proposed studies, we will test these hypotheses experimentally. Our specific aims are to: 1) identify the direct association of ECE expression with Purkinje fiber differentiation in the embryonic heart; 2) establish the inductive function of ET-signals during Purkinje fiber differentiation in vivo; and 3) determine links between the ET-signaling and CCS development in the mouse heart. Our basic strategies are to: 1) determine the timing and location of ECE expression during the CCS development at the both transcriptional and translational levels (Aim 1); 2) generate an ectopic site of activated ET-signaling within embryonic chick hearts (Aim 2); and 3) examine the CCS development in mice null for ET-related genes (Aim 3). The proposed studies will provide the foundation for understanding molecular mechanisms that induce differentiation of the CCS, and may also provide the basis for re-initiating CCS development in the adult heart in the future.